Limited proteolysis of fibrinogen by fibrinogenase from Echis multisquamatis venom

Area of Science:

• Biochemistry
• Proteomics
• Venom research

Background:

• Fibrinogenase from Echis multisquamatis venom primarily cleaves the BβArg42-Ala43 bond in fibrinogen.
• Limited hydrolysis of the Aα-chain was also observed previously.

Purpose of the Study:

• To investigate the accessibility of fibrinogenase hydrolysis sites in various fibrin(ogen) forms.
• To elucidate the cleavage pathway of Aα- and Bβ-chains in fibrinogen, monomeric fibrin desA, and polymeric fibrin desAB.

Main Methods:

• Western blot analysis using monoclonal antibodies specific to fibrinogen Bβ 26-42 and Aα 20-78.
• Incubation of fibrinogen and fibrin variants with purified fibrinogenase.

Main Results:

• The BβArg42-Ala43 bond is accessible in most fibrin(ogen) forms, except polymerized fibrin desAB, indicating BβN-domain involvement in protofibril formation.
• The Aα-chain is initially intact but undergoes hydrolysis in the αC-regions upon prolonged incubation, yielding ~30 and ~60 kDa fragments.
• Monomeric fibrin desA/desAB showed simultaneous Aα and Bβ chain hydrolysis, with Aα cleavage being more pronounced in polymeric fibrin.

Conclusions:

• The BβN-domain's role in protofibril formation renders the BβArg42-Ala43 site inaccessible in polymerized fibrin desAB.
• Fibrinogenase exhibits differential substrate specificity depending on the fibrin(ogen) form, with significant Aα-chain degradation occurring in specific fibrin states.